Combinatorial Electrochemical Synthesis

ABSTRACT

Abstract of Disclosure 
     An array of selectively addressible microelectrodes for combinatorial synthesis of complex polymers or alloys.

Cross Reference to Related Applications

[0001] This application is a continuation application of PCT ApplicationNo. PCT/US99/14459 A1, which claims priority to Provisional PatentApplication Serial No. 60/090,520, which applications are herebyincorporated for all purposes.

Background of Invention

[0002]Field of the Invention The present invention is generally relatedto the combinatorial syntheses of compounds, particularly of biologicalcompounds and metal alloys, on microelectrode arrays. The inventionrelates in particular to the combinatorial syntheses ofoligonucleotides, peptides and biologically active compounds onmicroelectrode arrays under electrochemical control.

[0003]Background of the Invention: The simultaneous synthesis of massivenumbers of different oligonucleotides, peptides, and biologically activecompounds has applications in the identification of new drugs, in themapping of genes, and in testing for interactions between biologicalmolecules. Arrays of different genes are applied, for example, indiagnosing various diseases, such as cancer and hereditary diseases, insequencing the human genome, and identification of drugs capable ofblocking bioconjugation reactions.

[0004] Conventional synthetic methods are time consuming and thestep-by-step preparation of individual potentially bioactive agentsconsumes the largest fraction of the capital invested in the developmentof new drugs. To address these problems, a variety of methods forcombinatorial syntheses has been developed. One commonly used methodinvolves the definition of pixels where a nucleotide is added to anexisting sequence through photochemical methods. Pixels for theoccurrence or the avoidance of a chemically synthetic step can bedefined by photolithographic methods. Alternatively, reactions in aparticular pixel can be driven by light when photochemically activereactants are used. Non-photochemical methods of combinatorial synthesesinvolve valved grids of microfluidic channels, such that a reactionoccurs in microreactors at the intersection points of the channels withopen valves. In general, these methods have resulted in pixels ormicroreactors that were as small as about 35 µm x 35 µm in theircross-sectional area. The syntheses involved small but still significantamounts of expensive reactants, typically more than a billion moleculesin each reaction in each pixel.

[0005] Michael Heller et.al, (U.S. Patent No. 5, 605,662) discloses aprocedure for producing arrays of selectively addressablemicroelectrodes. To transport and concentrate specific chargedoligonucleotides, an electrophoretic field is used. For example, apositive potential is applied to a specific microelectrode in order toattract a negatively charged oligonucleotide and to concentrate thecharged moiety at the site.

[0006] The present invention provides a method for parallel syntheseswhere a specific reaction is induced and controlled at themicroelectrode.

Summary of Invention

[0007] The present invention provides an electronic device and methodfor the combinatorial synthesis of biopolymers, alloys, andnon-stochiometric inorganic compounds. The device includes an array ofselectively addressable microelecrodes which preferably includes on itssurface a conductive matrix, which may be a redox polymer or hydrogel.The matrix further comprises functional reactive groups, such as amines,aldehydes, carboxyllic acids, active esters, and the like, useful forthe sequential addition of monomeric units to form polymeric compounds.For example, in one embodiment of the invention, the array includesamines in the matrix, to which nucleotides or oligonucleotides may besequentially added by chemical reaction.

[0008] In the method of the invention, a potential sufficient to inducea Faradaic reaction is selectively applied to each microelectrode toinduce binding of a reactant to one or more of the functional reactivegroups in the matrix. A complex compound is synthesized on themicroelectrode by repeated Faradaic reactions, i.e., by repeatedapplications of a potential sufficient to induce the desired reactions.

[0009] In an alternative embodiment, the method of the inventionincludes application of a potential to induce a Faradaic reaction andselectively deposit metals in layers onto a microelectrode. Optionally,a portion of one or more of the metal layers is etched or dissolved viasubsequent Faradaic reaction at the microelectrode. Heating, oxadation,sulfadation, or consolidation of two or more layers causes formation ofan alloy or non-stochiometric inorganic compound.

Brief Description of Drawings

[0010] Figure 1is a diagram showing the arrangement of the electrode orarray surface bearing the reactive groups as a monolayer or in apolymeric matrix. The electrode surface is schematically drawn as a flatsurface but the surface roughness (geometric area/actual area) may varybetween 1 and 1000.

[0011]Figure 2 is a flow diagrams for the syntheses of oligopeptides(poly amino acids, peptide nucleic acids or other oligopeptides). Thecoupling is carried out with carbodiimide in conjunction withN-hydroxysuccinimide. The solid bar connecting the COOH function to theelectrode is made of condensed amino acids. The amino acids may benatural amino acids or amino acids not found in nature.

[0012]Figure 3 is a flow diagram for the synthesis of oligonucleotides.The syntheses is modulated by the electrochemically generated protons.At potentials where protons are produced the local pH drops and theprotecting dimethoxytrityl (DMT) on the 5" end of the oligonucleotide iscleaved. The cleavage enables the extension of the oligonucleotide.

[0013]Figure 4 is a flow diagram showing the synthesis of anoligopeptide with an electrochemically pH modulated enzyme catalyzedstep.

[0014]Figure 5 is a schematic diagram showing the computerized automatedpotentiostatic and liquid delivery control to the microelectrode array.

Detailed Description

[0015]Microelectrode ArrayThe electronic device of the inventionincludes an array of selectively addressable microelectrodes. A matrixcomprising at least carbon (C) and hydrogen (H), and preferably capableof conducting electrons or holes, and also of conducting ions, isdisposed on a surface of the electrodes. The conductive matrix isdisposed on the microelectrode with a thickness greater than 3nanometers, and preferably in the range of 3 nanometers to 20 microns,most preferably with a thickness of 5 or more nanometers. Mostpreferably, the matrix comprises a redox polymer or redox hydrogel,having a molecular weight of more than 10⁴ daltons. Most preferably, theredox polymer or hydrogel comprises a fast redox couple, such ascomplexes of transition metals such as osmium, ruthenium, iron, copper,and cobalt.

[0016] The matrix is modified to include functional reactive groups,such as amines, aldehydes, carboxyllic acids, active esters, and thelike.

[0017] The arrays of electrodes on which the combinatorial syntheses iscarried out are usually large. Typically the number of electrodes in thearray is greater than 4, greater than 100, and is preferably greater1000 and it is most preferably greater than 10,000.

[0018] The longest dimension of a microelectrode is preferably less than100 µm, and is more preferably between 0.1 and 10 µm. The shape of amicroelectrode may be, for example, oval, rectangular or preferablycircular. Fine line electrodes, that are typically longer than 100 µmand have typical widths between 0.1 µm and 20 µm, can also be used. Itis not necessary that the microelectrode surface be flat. A metal orcarbon microelectrode surface may be, for example, concave or convexrelative to the plane of the surrounding insulating layer. Preferablyall the microelectrodes in the array are of the same dimensions and arespaced individually, or grouped in elements of the array, those elementsforming organized patterns. It is preferred that the pattern haverepeating elements and it is particularly preferred that the elementshave rotational and/or translational symmetry. An example of a preferredpattern is one where all electrodes except those at the periphery of thearray are equidistant from each other and have the same number ofnearest neighbors.

[0019] Typically it is desirable to space the electrodes at distancesgreater than 10 times the diameter or smallest dimension of themicroelectrodes and it is most preferred to space the electrodes atdistances greater than 20 times the diameter or smallest dimension ofthe microelectrodes. It is generally desirable to make the electrodessmall, so as to minimize the amount of material consumed in the reactiondriven at a subset of electrodes. Preferably fewer than one hundredmillion molecules are reacted at an electrode. It is more preferred thatfewer than one million be reacted and it is most preferred that fewerthan one hundred thousand be reacted. Reduction of electrode size alsoallows closer spacing of electrodes in an array, i.e. denser packing,meaning more electrodes per unit area. Typically the density ofmicroelectrodes is greater than 100/cm²; and most preferably it isgreater than 100,000/cm².

[0020] The electrodes are made of a conductor which is preferablynon-corroding and may comprise gold, carbon, tantalum, rutheniumdioxide, a conductive metal carbide, a conductive metal nitride, or aconductive metal oxide, or a Group VIII metal such as platinum, iridium,rhodium, rhenium, palladium or ruthenium. The electrodes are insulatedfrom each other by an insulator that may be organic or inorganic.Examples of inorganic insulators are silicon dioxide and silicon nitrideand insulating doped silicon. Examples of organic insulators arepolymers, such as poly(methyl methacrylate), polyimides, polyesters, orpolyamides. The substrate of the structure can be any material havingthe desired mechanical properties such as glass, ceramic, silicon,plastic, or a metal coated with an insulating film. The electricalcontacts to each element in the array may be in the plane of theelectrodes, or in the plane of the electrodes and in a second plane, orin multiple planes including or excluding the plane of the electrodes.Contacts to the electrodes may be formed individually to each electrode,to groups of electrodes, or to rows and columns of electrodes in a grid.

[0021] The elements of the array, i.e. its microelectrodes, can beconnected to potential or current sources by either hard wiring or bylight. Hard wiring means that the connection involves a continuouselectrical path through electrical conductors, particularly metallicconductors or carbon. Connection by light is possible when aphotoconductor is used, so that the illuminated areas conduct electronsor holes, while the non-illuminated areas do not conduct. Instead ofapplying an external potential or passing a current from an externalcurrent source, the potential at or the current passing through amicroelectrode of the array can also be photogenerated. For generationof a photopotential or a photocurrent it is preferred that themicroelectrode material be or comprise a semiconductor. Thesemiconductor may be an inorganic semiconductor, such as silicon or aIII-V compound. It may also be an organic compound, such as a polymericor non-polymeric organic compound used in light emitting diodes orphotodiodes or photovoltaic cells.

[0022] The electrochemical circuits require, in addition to themicroelectrodes on which the syntheses are carried out, known as workingelectrodes, at least one reference electrode and may require at leastone counter-electrode, that may or may not be the reference electrode.These added electrodes may be part of the array or may not be part ofit. The reference electrode may be a conventional reference electrodesuch as a saturated calomel or silver/silver chloride electrode or itmay be a pseudo-reference electrode consisting of a conducting materialin contact with the solution. The counter electrode can be a conductingmaterial with a surface area which is preferably to at least twice thesum of the combined surfaces area of the microelectrodes in the workingelectrode array. There may only be one reference electrode and onecounter electrode for the entire array.

[0023]Reactive groups on the microelectrode surface: The electrodesurface may be modified with reactive groups in a variety of waysdepending in the electrode material of choice. Two examples are shown inFigure 1. On the left, a matrix with remote reactive functions R, suchas a carboxylic acid functions, extending into the solution is shown. Onthe right, a polymeric gel/hydrogel support incorporating reactivefunctions R, such as carboxylic acid groups, attached to the back boneof the polymer is shown. Preferably the matrix should not be detachedunder the conditions of the reaction that is to be carried out. If thereaction is carried out under potentiostatic control, it is preferredthat the potential which is applied should affect only the reactivegroups or their reactions, not their attachment to the electrode.

[0024] The conducting microelectrode, or group of microelectrodes, orthe surface of the entire microelectrode array, including the surface ofthe insulating material between the electrodes, may be modified with amatrix. This matrix can, for example, be a matrix having affinity for areactant, such as a polycation when the reactant is a nucleotide or anucleotide derivative which is an anion. It can also be a matrixcomprising a redox polymer or a redox hydrogel, the polymer or gelcontaining attached redox centers. When a redox hydrogel or conductivepolymer is used, it is preferred that only the conducting region of thearray be covered.

[0025]Method of the InventionIn the method of the invention, an array ofdesired chemical compounds is selectively synthesized on selectivelyaddressable microelectrodes. A potential sufficient to cause a Faradaicreaction in the immediate vicinity of the microelectrode is selectivelyapplied to each microelectrode to induce binding of a first reactant toone or more funcional reactive groups present in the matrix disposed onthe microelectrode. After binding of a first reactant to a functionalreactive group in the matrix, the selective application of potential isrepeated to cause a second Faradaic reaction and induce binding of asecond reactant to the first bound reactant. This procedure is repeateda sufficient number of times to synthesize a desired compound on themicroelectrode, thus forming an array of individual compounds.

[0026] The reactants may be, for example, nucleotides, amino acids,chemical moieties, or other molecular subunits such as oligonucleotidesor peptides that are induced to bind amines, aldehydes, carboxylicacids, active esters, and the present in the matrix.

[0027] The Faradaic reaction preferably causes a chemcial change in oneor more of the fuctional reactive groups in the matrix, in a reactantsupplied to the microelectrode, for example in a solution bathing theelectrode, or in a further chemical species present in the solution. Thechemical change can result in a change in pH, in the oxidation orreduction of a functional reactive group on the matrix, of the suppliedreactant, or of a further chemical species in the reaction solution.

[0028]Control through a faradaic process: A Faradaic current is acurrent that passes when a species is electrooxidized or electroreducedat an electrode. This current is contrasted with a capacitive orpolarization current where a current will flow for a period of time whena potential is applied without an electrooxidation or electroreductionreaction taking place.

[0029] The capacitive or polarization current flows as a result of theredistribution of ions in the solution (or film) near the electrode. Theelectrophoretic process, where a macromolecular polyelectrolyte migratesto an electrode is such a non-Faradaic process. After some time thiscurrent stops because of the accumulation of cations near a negativeelectrode or of anions near a positive electrode.

[0030] Upon flow of a Faradaic current, a substance is electrolyzed. Atleast one species in the immediate vicinity of the microelectrode (thatis, the distance from the microelectrode where the microelectrode cancause redox reactions) is electrooxidized and at least one species iselectroreduced. As a result, there is a net change in the chemicalcomposition in the immediate vicinity of the microelectrode, not a merere-distribution of ions in the cell.

[0031] Faradaic reactions, unlike capacitive or polarization processes,take place only when sufficient potential is applied to an electrode.This is well known form any text in electrochemistry or even physicalchemistry. The threshold potential is the formal half cell potential forthe reaction driven. When the reaction is reversible, then the potentialis the thermodynamic potential, - ΔG/(nF), where ΔG is the change in theGibbs free energy, n is the number of electrons gained or lost by thereduced or oxidized species and F is Faraday"s constant. For example,the threshold potential for electrolysis of water is 1.23 V (measuredrelative the standard hydrogen electrode). A Faradaic current resultingin water electrolysis flows only when this potential is exceeded.

[0032] Acid is produced by electrolysis of water at a half cellpotential depending on the pH and temperature. The higher the pH, themore reducing (negative) this potential is, shifting at 25^(o)C by 59 mVfor each pH unit (when the partial pressure of hydrogen isfixed).Reversible potentials are listed in most handbooks of chemistry,such as the Handbook of Chemistry and Physics.

[0033] The Faradaic reaction need not be reversible. For example, whenascorbic acid is electrooxidized the reaction is irreversible. Thecomposition of the solution within the cell changes in the reaction. Thecompound is not only redistributed but converted.

[0034] The local concentration of ions, particularly of protons, andthus the local pH can be controlled through controlling the currentdensity passing through a particular electrode, which can be in turn,controlled by the applied potential. Upon electrooxidation, protons areusually released and upon electroreduction protons are usually consumed.

[0035] The reactant in the faradaic process can be oxygen, the solventitself, e.g. water; a readily electrooxidizable organic compound such asascorbic acid, or a readily electroreducible organic compound, such asbenzoquinone. The reactant can also be an inorganic or metal-organicion, such as a complex of iron, cobalt, ruthenium, osmium or copper. Forexample, the reactant can be a bipyridine complex of ruthenium or osmiumor cobalt; a cyanide complex of iron; or a metallocene derivative, suchas a ferrocene derivative.

[0036] The metal of the electrode or a metal ion may be reduced oroxidized to an oxidation state which is suitable for the modulation ofthe chemical reaction at the surface of the electrode. The reactant mayalso be an organic molecule which can be electroreduced orelectrooxidized to a species which chelates with a dissolved metal ion.The electrochemically formed chelator reduces the local free metal ionconcentration at the electrode surface.

[0037]Control through application of a potential without the occurrenceof a substantial faradaic (electroreduction or electrooxidation)reaction:As taught in Heller et.al., U.S. Patent No. 5,605,662, thelocal concentration of ions at a particular microelectrode or group ofmicroelectrodes may be controlled by a capacitive process, such as aprocess of attracting cations, repelling cations, attracting anions orrepelling anions. Examples of the anions attracted or repelled arehydrated protons (H⁺), hydroxide anions (OH⁻) and Zn²⁺, Mg²⁺, Ca²⁺ orCu²⁺ ions.

[0038] The potential applied to the electrode for eletrophoretictransport and concentration of a charged species is mirrored by the ionsin the solution. Thus a positive applied potential (relative to thepotential of zero charge (PZC) of the particular metal in the solution)will draw anions from the bulk solution, causing a local build up ofanions at the electrode interface relative to the bulk solution. Theattracted anions will replace cations at the surface. The pH or ionconcentration in the proximity of an electrode can be controlled throughthe potential applied, and/or the buffer added to the solution and/orthe concentration of the relevant ion in the solution.

[0039] The number of electrodes in the array is greater than tenthousand and the dimension of each microelectrode is 10 µm or smaller.The use of microelectrodes ensures that mass transport of species to andaway from the electrode surface is efficient. Concentrationpolarization, which is the change in concentration of a reactant near anoperating electrode, and is important in defining the necessary spacingof the electrodes, is also reduced when microelectrodes are used. Thusdenser electrode arrays can be made.

[0040] The extent of concentration polarization, meaning the distance towhich a concentration change extends from a particular electrode dependson the diffusion coefficient of the reactant, the dimension of themicroelectrode and the applied potential. The occurrence or preventionof a reaction at a particular electrode is controlled by applying apotential to, or passing a current through, the electrode at which theoccurrence of the reaction is desired or in which the occurrence of thereaction is to be prevented. The application of a potential induces afaradaic or non-faradaic process, depending upon the specific potentialapplied and the species present in the vicinity of the electrode, whichchanges the concentration of at least one ion or molecule relative tothe bulk solution.

[0041] The concentration of an ion near a particular microelectrode canbe controlled through a faradaic process, such as an electroreductionreaction or an electrooxidation reaction. It is well known that thepotential where an electrooxidation or electroreduction reaction takesplace depends on the presence of a particular electrooxidizable orelectroreducible species in the solution. In general the local pH at andnear the electrode surface is increased when an electroreductionreaction is taking place and is decreased when an electrooxidationreaction is taking place. Usually the higher the current density thegreater the change in local pH. The magnitude of the change also dependson the concentration of buffering agents, decreasing when the bufferconcentration is raised. For example, the local pH may be increased at amicroelectrode by the electroreduction of oxygen, a reaction whereprotons are consumed.

[0042] The pH can be decreased locally by the electrooxidation of waterto hydrogen peroxide or to oxygen, or by the electrooxidation of asolute, such as ascorbate ion, that takes place at a less oxidizingpotential than the potential for electrooxidation of water. In thesereactions protons are released. The extent of pH change may becontrolled by the potential applied or by the buffering capacity of thesolution. In summary the local concentration of a particular speciesnear a particular electrode is controlled through the potential appliedor the current passed, or by the concentration and nature of an addedbuffer.

[0043] There are different ways through which the local concentrationsof ions at a microelectrode surface may control the occurrence ornon-occurrence of a reaction. For example, a reactive species such as anO-acylisourea or an N-hydroxysuccinimide ester does not react with anamine in an acid environment, where the amine is protonated. However, atneutral or slightly basic pH the reaction with the amine does take placeand an amide is formed. Through controlling the local concentration ofprotons, or of other ions, or of a molecule such as ascorbate which isrequired for a reaction to take place at a defined potential, it is alsopossible to modulate the activity of enzymes that catalyze either theformation or the breakage of chemical bonds, for example bonds formed incondensation or hydrolysis reactions. There are numerous well documentedexamples, found in textbooks of biochemistry, where an enzyme is activeonly in a well defined pH range. Also some enzyme-catalyzed reactionsrequire the presence of a particular ion, such as Zn²⁺ or Mg²⁺, or Ca²⁺;others can be reversibly or irreversibly inhibited by the presence ofother ions, e.g. Cu²⁺.

[0044] Examples of non-faradaic processes whereby the localconcentration of an ion is changed include local increase of theconcentration of a cation when a negative potential is applied to anelectrode; increase in the local concentration of an anion when apositive potential is applied; local decrease in concentration of acation when a positive potential is applied; and local decrease in theconcentration of an anion when a negative potential is applied. Theabove four processes do not require the occurrence of an electrochemicalreaction.

[0045] Examples of relevant reactions known to take place in aparticular pH domain: Examples of such reactions include the synthesesof amides such as those of oligopeptides including peptides, proteinsand in peptide nucleic acids via a carbodiimide involving reaction;syntheses involving N-hydroxysuccinimide esters; syntheses involvingimidates; and synthesis involving polyphosphates. Figure 2 shows anexample of a flow diagram of a synthesis utilizing a carbodiimide andNfor the formation of a peptide bond on an electrode surface.

[0046] When the objective is not to make amides but other compounds,other active reactants may be used. For example an epoxide may be usedfor a reaction with an amine; or an alkyl halide, particularly an alkyliodide or an alkyl bromide, for a reaction of an amine. The reactiontakes place in neutral or basic solutions, but not in acid ones. Controlcan be either by adjusting the local pH through a faradaic or anon-faradaic process such that the reaction proceeds, or throughinhibiting the reaction through adjusting the local pH such that thereaction is prevented.

[0047] For the electrically controlled synthesis of oligonucleotides,the well-known phosphoramidite method can be applied. Figure 3 shows theflow diagram of a current or a potential controllable synthetic route,based on the phosphoramidite method and involving a dimethoxytrityl(DMT) protecting group on the 5" terminus of the reacting base. Whenprotons are generated at the microelectrode, the protecting group on the5" is removed. This removal or de-protection allows the extension of theoligonucleotide once the cycle is repeated. Because the coupling step isalso pH dependent, the coupling can also be modulated by an appliedpotential or a current passed through the electrode. Also the iodinerequired for the oxidation step may be electrochemically generated whenthe solution comprises iodide ions. Any unreacted oligonucleotide iscapped by acylation with acetic anhydride to avoid extension of anyundesired sequences. This capping is not shown in Figure 3.

[0048]Enzyme Catalyzed Reactions: There are families of enzymes known tocatalyze the hydrolysis or formation of amides in peptides, proteins,and protein nucleic acids and of phosphate ester links inoligonucleotides or DNA and of glycosidic linkages in oligosaccharides.The families of these enzymes include, for example, kinases, peptidases,proteolytic enzymes and hydrolases, transferases, ligases. The enzymes"activity can be controlled by the local adjustment of the pH. Also theactivity of some enzymes may be enhanced by the local electrochemicalreaction or process (meaning application of potential or passage ofcurrent) increase in the concentration of ions such as Mg²⁺, Ca²⁺ , orZn²⁺, or may be decreased by electrochemically increasing the localconcentration of ions such as Cu²⁺. The enzyme may cause the addition,through formation of a covalent bond, of a dissolved species in thesolution to which the array is exposed; or it may be such that itcleaves a terminal function on a molecule already on the electrode so asto enable a reaction at that particular electrode; or it may causehydrolysis of a functional group of a molecule on an electrode.

[0049] A schematic diagram of the steps involved in the enzyme catalyzedreaction is shown in figure 4. The method described is the step by stepsynthesis of a peptide nucleic acid modulated by a non-specific amidase,particularly acrylamide amidohydrolase from Pseudomonas aeruginosa.Enrichment of the zone near the microelectrode in protons prevents theenzymatic cleavage of the terminal amide and therefore the subsequentcarbodiimide or N-hydroxysuccinimide ester (NHS) utilizing condensationreaction whereby an amino acid is added to the peptide, peptide nucleicacid or protein on the electrode.Automated system: The system may beautomated by integrating a computer to control the flow of liquidcontaining reactants by a series of valves and also the potentiostat asshown in figure 5.

[0050] For example, a series of different oligonucleotides can be formedon an array of electrodes using the apparatus illustrated in figure 5and the process illustrated in figure 3. An array of carbon or metallicelectrodes is formed. Each of the electrodes is coated with a couplingspecies. The coupling species includes a reactive functionality that isinitially capped with a protective group. The array is placed within aflow device that is coupled to a valve, which is, in turn, coupled tofour reservoirs of molecular subunits, corresponding to the fourdifferent bases of DNA; adenine, guanine, thymine, and cytosine. Each ofthe molecular subunits includes a first reactive functionality forcoupling to a deprotected reactive functionality of the coupling speciesor a previously deposited molecular subunit. Each of the molecularsubunits also includes a second reactive functionality that is initiallycapped by a protecting group.

[0051] In operation, the valve is directed to open and allow one of thefour solutions of molecular subunits to flow into contact with thearray. Each electrode of the array is individually coupled to apotentiostat, typically, under computer-control. A potential isselectively applied to (or, alternatively, a current is passed through)those electrodes at which the particular molecular subunit is to bedeposited. The electrical potential (or current) causes a change in theconcentration of an anionic or cationic species (e.g., a change in pH)that leads to the removal of the protecting group on the couplingspecies and results in a reaction of the reactive subunit on thecoupling species and the first reactive functionality of the molecularsubunit.

[0052] The solution containing the first molecular subunit is thenremoved and the valve is directed to open and allow a second solutionwith a different molecular subunit to flow into contact with the array.Again, a potential is selectively applied to those electrodes at whichthis particular molecular subunit is to be attached, including thoseelectrode at which this second molecular subunit is to be attached tothe first molecular subunit. The application of the potential results inthe removal of the protecting group from the reactive group of thecoupling species or the second reactive group of the previously coupledmolecular subunit. This procedure is repeated until the desiredoligonucleotide sequences are all formed on the array of electrodes.

[0053] As an example, a four electrode array can be formed with thefollowing oligonucleotides on the electrodes:

[0054] Electrode 1 - AGTC

[0055] Electrode 2 - ATGC

[0056] Electrode 3 - GTGC

[0057] Electrode 4 - TGCA

[0058] One exemplary process includes the steps in the following table:

[0059] Other sequences of steps could also be used to obtain the samearray of electrodes. In addition, the same principle can be used to formother molecules, such as peptides or proteins, on electrodes thatutilize a small set of subunits, such as amino acids.

[0060]Combinatorial Synthesis of Inorganic MaterialsIn the combinatorialsyntheses of inorganic compounds or organic compounds having inorganicbackbones a potential is applied to a microelectrode of the array or acurrent is passed through an electrode of the array, such that the localpH is changed or the local concentration of another ion is changed. Whenthe reactive material on the microelectrode is a metal or metal oxide,then typically a reduction in pH, the application of a positivepotential, or the occurrence of a local electrooxidation reaction canaccelerate the dissolution of the oxide or the metal. For example,metals, such as zinc or aluminum, or an oxide of such metals are morerapidly dissolved by a local rise in pH. The rate of removal is adjustedthrough local control of the pH, the potential, or the current. Thus byvarying in a gradual manner the potential or the current density theamount of residual material residing on an electrode after partialstripping of a layer can be increased or decreased. Similarly by drivingan electroreduction reaction, whereupon the pH is increased, the amountof residual material may be increased when the film is in an etchingsolution, such as an acid solution.

[0061] The removal and/or deposition of inorganic material can be usefulin a variety of circumstances, including, for example, the combinatorialformation of non-stoichiometric materials. These materials are oftentested for various properties, including, for example, fluorescencewavelength, fluorescence quantum yield, magnetic properties, anddielectric constant. It is often useful to test a range of differentnon-stoichiometric combinations. A material is "non-stoichiometric" ifthe composition can not be expressed as a chemical formula using numbersor integers of smaller than five.

[0062] One method for forming a range of non-stoichiometric combinationsincludes forming an array of metal electrodes 102 on a substrate 114, asshown in Figure 1. By selectively applying different potentials or byvarying the duration of the applied potential to the electrodes of thearray, different amounts of the electrode can be removed.

[0063] The rate of removal is determined, at least in part, by the localconcentration of other anionic or cationic species around the electrode.This local concentration is modified by the potential applied to theelectrode. For example, the pH can be altered by applying a potential,which can then cause the dissolution of the a portion of the metalelectrode. The amount of the metal electrode that is removed depends, atleast in part, on the potential, the current density through theelectrode, and the period of time the potential is applied or thecurrent is passed. By varying any of these parameters across the array,the amount of material removed varies.

[0064] Next, a second metal can be deposited on the electrodes and theprocess of applying a potential is repeated. This can continue for anynumber of metal deposition steps. Alternatively, the metal may beselectively deposited by applying a potential that causeselectroreduction of metal cations from a solution. The amount of metalthat is deposited depends on the potential, the current density throughthe electrode, and the period of time the potential is applied. In yetanother embodiment, porous films of metal compounds, such as, forexample, metal oxides, can be formed on the electrode and a portion ofthe metal compounds can be selectively removed by applying a potential.

[0065] After all of the metal and/or metal compound depositions areperformed, and an array of electrode structures with differentcombinations of metals and metal compounds are formed, the metal and/ormetal compounds can be converted into a desired alloy or compound by,for example, heating, oxidation, sulfidation, other chemical reactions,or consolidation of the various layers.

[0066] This method can be useful for the formation of an array ofdifferent non-stoichiometric combinations of materials, each combinationbeing determined, at least in part, by the particular potentials anddurations applied to the electrode during each step.

[0067] Yet another means of control involves increasing the localconcentration of an anion with which the metal of the electrode mayreact to form a soluble complex. For example, gold is known to react atmildly oxidizing potentials with dissolved chloride ions. If the localconcentration of chloride ions is increased by applying a positivepotential, then the rate of the dissolution of the gold will alsoincrease, even if the local pH is unchanged.

[0068] In the hydrolytic reaction and precipitation of solution phaseinorganic or mixed organic-inorganic compounds, such as halides oralkoxides of Si, Ti, Zr or Al, the nature and reactivity of the productmay be controlled at an electrode of the array through the local changein pH. The formation of the solution phase inorganic or mixedorganic-inorganic compounds, such as a polymer, can be controlled byapplication or non-application of a potential to each electrode in thearray. In addition, the structure of the compound may, at least in somecases, be dependent on the potential applied at the electrode. Forexample, the polymer formed upon hydrolysis of a silicone precursor,such as methyl trimethoxysilane depends on the local pH. Ladder-typesilsesquioxanes are often formed at higher pH.

[0069]Exemplary array and usePVP-Os-NH₂: A redox polymer comprising apoly (4-vinyl pyridinie) backbone here about 0.10 of the pyridines arecomplexed with [Os(bpy)₂Cl]^(+/2+); and about 20% are reacted with2-bromoethylamine and are thereby quarternized ( bpy is 2,2'-bipyridine;PEGDGE is poly(ethylene glycol)diglycidyl ether , molecular weight400-600; SCE is standard calomel electrode potential.

[0070] An array of four gold electrodes is produced on a quartz plate,each electrode spaced a distance of 100 micrometers from the other, andinsulated from eachother. Each gold electrode (2 micrometers indiameter) is connected to a contact pad. A solution of PVP-OS-NH₂ (10mg/ml) is incubated with PEGDGE (1 mg/ml) for two hours at 37^(o)C, pH7.

[0071] The electrodes are placed into contact with the PVP-OS-NH₂ /PEGDGE solution, and a potential of -0.6 Volts (SCE) is applied for 10minutes to electrophoretically deposit and crosslink the redox polymer.The solution is replaced with a solution of oligonucleotides (DNA orRNA) (10 mg/ml), and the potential is reversed to +0.6 Volts (SCE) forten minutes. The array is then placed into a solution of ascorbic acidand sodium ascorbate (pH 7.5), total concentration 0.5mM. A potential of0.4 volts (SCE) is then applied to two of the electrodes of the array.

[0072] A drop of micrococcal nuclease (300,000 U/ml) from Staphlococcusaureus is added to the array. DNA or RNA is hydrolized only at thoseelectrodes to which no potential is applied, and is not hydrolyzed atthose electrodes at those electrodes to which a potential is applied.

[0073] The foregoing description contains numerous references topublications and patents, each of which is hereby incorporated byreference, for all purposes, as if fully set forth.

Claims
 28. A device comprising: a) a plurality of selectively adressablemicroelectrodes; b) a conductive matrix disposed on each microelectrode,the matrix comprising carbon, hydrogen and functional reactive groups,wherein the functional reactive groups are activated or deactivated byapplying a current or a potential to the conductive matrix; and c) asource of current or potential arranged and configured to selectivelyprovide a current or voltage to each microelectrode, wherein each of theselectively addressable microelectrodes has a smallest lateraldimension, and wherein each microelectrode is separated from othermicroelectrodes of the device by a distance of at least ten times thesmallest lateral dimension of the microelectrode.
 29. The deviceaccording to claim 28, wherein the smallest lateral dimension is adiameter.
 30. The device according to claim 28, wherein the smallestlateral dimension is measured from one edge of the microelectrode to anopposite edge of the microelectrode.
 31. The device according to claim28, wherein each microelectrode is separated from other microelectrodesof the device by a distance of at least twenty times the smallestlateral dimension of the electrode.
 32. The device according to claim28, wherein the smallest lateral dimension of one or more of themicroelectrodes is less than 100 μm.
 33. The device according to claim32, wherein the smallest lateral dimension of each of themicroelectrodes of the device is the same.
 34. The device according toclaim 28, wherein the smallest lateral dimension of one or more of themicroelectrodes is 0.1 μm to 1 μm.
 35. The device according to claim 34,wherein the smallest lateral dimension of each of the microelectrodes isthe same.
 36. The device according to claim 28, wherein the conductivematrix comprises a redox polymer.
 37. The device according to claim 36,wherein the redox polymer comprises a transition metal, and wherein thetransition metal is osmium, ruthenium, iron, copper or cobalt.
 38. Thedevice according to claim 36 wherein the redox polymer comprises ahydrogel.
 39. The device according to claim 28 wherein the conductivematrix comprises a polycation.
 40. The device according to claim 28wherein the conductive matrix has a thickness of 3 nm to 20 μm.
 41. Thedevice according to claim 28, wherein the functional reactive groups areindependently selected from amines, aldehydes, carboxylic acids, oractive esters.
 42. The device according to claim 28 further comprisingone or more reference electrodes.
 43. The device according to claim 28further comprising one or more counter-electrodes.
 44. A method forselective synthesis of an array of compounds, the method comprisingsteps of: a) providing a device comprising: (i) a plurality ofselectively addressable microelectrodes; (ii) a conductive matrixdisposed on each microelectrode, the matrix comprising carbon, hydrogenand functional reactive groups, wherein the functional reactive groupsare activated or deactivated by applying a current or a potential to theconductive matrix; and (iii) a source of current or potential configuredand arranged to selectively provide a current or voltage to eachmicroelectrode, wherein each of the selectively addressablemicroelectrodes has a smallest lateral dimension, and wherein eachmicroelectrode is separated from other microelectrodes of the device bya distance of at least ten times the smallest lateral dimension of themicroelectrode; b) providing a first reactant; and c) selectivelyapplying to one or more microelectrodes a current or potentialsufficient to cause a faradaic reaction in the immediate vicinity of theone or more microelectrodes to induce binding of the first reactant tothe conductive matrix.
 45. The method according to claim 44, furthercomprising repeating the step of selectively applying to one or moremicroelectrodes a potential sufficient to cause a faradaic reaction inthe immediate vicinity of the one or more microelectrodes to inducebinding of an additional reactant to form an array of compounds.
 46. Themethod according to claim 44, further comprising selectively applying toone or more microelectrodes a potential sufficient to cause a faradaicreaction in the immediate vicinity of the one or more microelectrodes toinduce binding of a second reactant to the first reactant.
 47. Themethod according to claim 44, wherein the faradaic reaction causes achemical change in one or more of the functional reactive groups, thereactant, or a chemical species, in the immediate vicinity of themicroelectrode.
 48. The method according to claim 47, wherein thechemical change is a change in ionic concentration or an oxidation or areduction of the functional reactive groups, the reactant, or thechemical species.
 49. The method according to claim 48 wherein thechange in ionic concentration is a change in pH.
 50. The methodaccording to claim 48, further comprising providing an enzyme, whereinthe chemical change is a change in ionic concentration, and whereinadjustment of the ionic concentration in the immediate vicinity of themicroelectrode modulates activity of the enzyme in the immediatevicinity of the microelectrode.
 51. The method according to claim 44wherein the reactant comprises a nucleotide.
 52. The method according toclaim 44 wherein the reactant comprises an amino acid.
 53. The methodaccording to claim 44 wherein the reactant comprises an organiccompound, an inorganic compound or a metal-organic ion.
 54. The methodaccording to claim 53 wherein the organic compound is ascorbic acid orbenzoquinone.
 55. The method according to claim 53 wherein the inorganiccompound is iron, cobalt, ruthenium, osmium or copper.
 56. The methodaccording to claim 44 wherein the method comprises: a) providing adevice comprising: (i) a plurality of selectively addressablemicroelectrodes; (ii) a redox polymer comprising poly(4-vinyl pyridine),osmium and amine reactive groups; and (iii) a source of current orpotential configured and arranged to selectively apply a current orvoltage to each microelectrode; b) providing a first nucleotide; and c)selectively applying to one or more microelectrodes a current orpotential sufficient to cause a faradaic reaction in the immediatevicinity of the one or more microelectrodes to induce binding of thefirst nucleotide to the redox polymer.
 57. The method according to claim56 further comprising: d) providing a second nucleotide; and e)selectively applying to one or more microelectrodes a current orpotential sufficient to cause a faradaic reaction in the immediatevicinity of the one or more microelectrodes to induce binding of thesecond nucleotide to the redox polymer or to one or more of the firstnucleotides.
 58. The method according to claim 56 wherein the step ofselectively applying to one or more microelectrodes a current orpotential sufficient to cause a faradaic reaction in the immediatevicinity of the one or more microelectrodes induces binding of the firstnucleotide to one or more of the amine reactive groups.
 59. A method forselective synthesis of an array of compounds, the method comprisingsteps of: a) providing a device comprising: (i) a plurality ofselectively addressable microelectrodes; (ii) a conductive matrixdisposed on each microelectrode, the matrix comprising carbon, hydrogenand functional reactive groups, wherein the functional reactive groupsare activated or deactivated by applying a current or a potential to theconductive matrix; and (iii) a source of current or potential providinga selective current or voltage to each microelectrode, wherein each ofthe selectively addressable microelectrodes has a smallest lateraldimension, and wherein each microelectrode is separated from othermicroelectrodes of the device by a distance of at least ten times thesmallest lateral dimension of the microelectrode; and b) selectivelyapplying to one or more microelectrodes a current or potentialsufficient to cause a faradaic reaction in the immediate vicinity of themicroelectrode to induce deposit of a metal onto the microelectrode. 60.The method according to claim 59 further comprising repeating the stepof selectively applying to one or more microelectrodes a potentialsufficient to cause a faradaic reaction in the immediate vicinity of themicroelectrode to induce deposit of a second metal onto themicroelectrode to synthesize a non-stoichiometric inorganic compound ormetal alloy on the microelectrode.
 61. The method according to claim 59further comprising the step of inducing etching or dissolution of aportion of one or more metals deposited onto the microelectrode.
 62. Themethod according to claim 61 further comprising reacting by heating,oxidation, sulfidation, or consolidation to form an alloy ornon-stoichiometric inorganic compound.
 63. A method for selectivesynthesis of an array of compounds, the method comprising steps of: a)providing a device comprising: (i) a plurality of selectivelyaddressable microelectrodes; (ii) a conductive matrix disposed on eachmicroelectrode, the matrix comprising carbon, hydrogen and functionalreactive groups; and (iii) a source of current or potential configuredand arranged to selectively provide a current or voltage to eachmicroelectrode, wherein each of the selectively addressablemicroelectrodes has a smallest lateral dimension, and wherein eachmicroelectrode is separated from other microelectrodes of the device bya distance of at least ten times the smallest lateral dimension of themicroelectrode; b) providing a first reactant; c) providing an enzyme;and d) selectively applying to one or more microelectrodes a current orpotential sufficient to cause a faradaic reaction in the immediatevicinity of the one or more microelectrodes to change ionicconcentration in the immediate vicinity of the one or moremicroelectrodes, wherein change of the ionic concentration in theimmediate vicinity of the one or more microelectrodes modulates activityof the enzyme, and wherein the enzyme catalyzes reaction of the firstreactant with the functional reactive groups.